US20040259151A1 - Functional surface display of polypeptides - Google Patents

Functional surface display of polypeptides Download PDF

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US20040259151A1
US20040259151A1 US10/469,723 US46972303A US2004259151A1 US 20040259151 A1 US20040259151 A1 US 20040259151A1 US 46972303 A US46972303 A US 46972303A US 2004259151 A1 US2004259151 A1 US 2004259151A1
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host cell
adx
protein
polypeptide
recombinant polypeptide
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Joachim Jose
Frank Hannemann
Rita Bernhardt
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Zyrus Beteiligungs GmbH and Co Patente I KG
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UNIVERSITAT DES SAARLANDES WISSENS-UND TECHNOLOGIETRANSFER GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • the present invention relates to a method for the display of recombinant functional polypeptides containing a prosthetic group and/or a plurality of subunits on the surface of a host cell using the transporter domain of an autotransporter.
  • an artificial precursor must be constructed by genetic engineering, consisting of a signal peptide, the recombinant passenger, the ⁇ -barrel and a linking region in between, which is needed to achieve full surface access (21).
  • the AIDA-I autotransporter was successfully used in this way for efficient surface display of various passenger domains (15,16,27,28).
  • the autotransporter mediated surface display has been restricted to monomeric proteins that were devoid of any non-proteinaceous cofactors. This can be due to the fact that a polypeptide chain to be transported by the autotransporter pathway must be in an relaxed, unfolded conformation.
  • adrenodoxin belongs to the [2Fe-2S] ferredoxins, a family of small acidic iron-sulfur proteins, which can be found in bacteria, plants, and animals (29). It plays an essential role in electron transport from adrenodoxin reductase (AdR) to mitochondrial cytochromes P450, which are involved in the synthesis of steroid hormones (FIG. 1) (30). Moreover, cytochromes P450 play an essential role in the biosynthesis of prostaglandins or secondary metabolites of plants and microorganisms, as well as in the detoxification of a wide range of foreign compounds as drugs or chemical pollutants (41).
  • the iron-sulfur cluster of Adx is coordinated by four sulfur atoms from side chains of four of its five cysteine residues (31).
  • Bovine adrenodoxin is encoded by a nuclear gene, synthesized in the cytoplasm and processed upon mitochondrial uptake. The mechanism of iron-sulfur cluster incorporation is still not clear, however, during heterologous expression in E. coli, it can be assembled in the cytoplasm as well as in the periplasm (32).
  • a fusion protein comprised of a signal peptide, a monomeric Adx and the transporter domain of AIDA-I and its efficient and stable surface display.
  • the iron-sulfur cluster could be incorporated by a one step procedure, yielding functional Adx.
  • autodisplay can be applied to proteins that contain inorganic cofactors and that these proteins are freely accessible at the cell surface, even for large ligands or partner proteins as adrenodoxin reductase or cytochromes P450.
  • a first aspect of the present invention is a method for displaying a recombinant polypeptide containing a prosthetic group on the surface of a host cell comprising the steps:
  • nucleic acid fusion operatively linked with an expression control sequence; said nucleic acid fusion comprising:
  • polypeptides e.g. enzymes requiring for functionality a prosthetic group
  • the prosthetic group may be combined with the recombinant polypeptide on the surface of the host cell, on a membrane preparation derived from the host cell or after cleavage of the recombinant polypeptide from the host cell or a membrane preparation thereof.
  • the procedure wherein the prosthetic group is combined with the recombinant polypeptide may comprise thermal treatment, e.g. heating and/or chemical treatment, e.g. reduction, oxidation and/or pH adjustment. It should be noted that also a plurality of prosthetic groups which may be identical or different may be combined with the recombinant polypeptide.
  • the prosthetic group may be selected from any non-proteinaceous component which is known to function as a prosthetic group.
  • the prosthetic group may comprise an inorganic component such as a metal which may be present as a metal ion.
  • the metal may be selected from heavy metals such as cobalt, nickel, manganese, copper and iron.
  • Preferred examples of prosthetic groups are [2Fe-2S] clusters, [4Fe-4S] clusters and metal porphyrin, e.g. heme groups.
  • a prosthetic group may be selected which comprises an organic component, e.g. a coenzyme.
  • organic component e.g. a coenzyme.
  • prosthetic groups are flavin containing groups, e.g. FMN or FAD, nicotin containing groups, e.g. NAD, NADH, NADP or NADPH, biotin, ⁇ 2-microglobulin, thiamine pyrophosphate, coenzyme A, pyridoxal phosphate, coenzyme B12, biocytine, tetrahydrofolate and lipoic acid.
  • a further embodiment of the present invention relates to a method for displaying a recombinant multimeric polypeptide on the surface of a host cell comprising the steps:
  • nucleic acid fusion operatively linked with an expression control sequence said nucleic acid fusion comprising:
  • the multimeric recombinant polypeptide may be a homodimer, i.e. a polypeptide consisting of two identical subunits or a homomultimer, i.e. a polypeptide consisting of three or more identical subunits.
  • the multimeric recombinant polypeptide may be a heterodimer, i.e. a polypeptide consisting of two different subunits or a heteromultimer consisting of three or more subunits wherein at least two of these subunits are different.
  • the multimeric polypeptide is comprised of a plurality of subunits which form a “single” multimeric polypeptide or a complex of a plurality of functionally associated polypeptides which may in turn be monomeric and/or multimeric polypeptides. It should be noted that at least one subunit of the multimeric recombinant protein may contain at least one prosthetic group. Further, is should be noted that the nucleic acid fusion may encode a plurality of polypeptide subunits as a polypeptide fusion which when presented on the cell surface forms a functional multimeric polypeptide.
  • Homodimers or homomultimers may be formed by a spontaneous association of several identical polypeptide subunits displayed on the host cell membrane.
  • Heterodimers or heteromultimers may be formed by a spontaneous association of several different polypeptide subunits displayed on the host cell membrane.
  • a multimeric recombinant polypeptide may be formed by an association of at least one polypeptide subunit displayed on the host cell membrane and at least one soluble polypeptide subunit added to the host cell membrane.
  • the added subunit may be identical to the displayed subunit or be different therefrom.
  • the host cell used in the method of the present invention is preferably a bacterium, more preferably a gram-negative bacterium, particularly an enterobacterium such as E. coli.
  • a host cell particularly a host bacterium is provided which is transformed with at least one nucleic acid fusion operatively linked with an expression control sequence, i.e. a promoter and optionally further sequences required for gene expression in the respective host cell.
  • the nucleic acid fusion is located on a recombinant vector, e.g. a plasmid vector.
  • these nucleic acid fusions may be located on a single vector or on a plurality of compatible vectors.
  • the nucleic acid fusion comprises (i) a portion encoding a signal peptide, preferably a portion coding for a gram-negative signal peptide allowing for transport into the periplasm through the inner cell membrane. Further, the nucleic acid fusion comprises (ii) a portion encoding the recombinant polypeptide to be displayed and (iii) optionally a portion encoding a protease recognition site, which may be a recognition site for an intrinsic protease, i.e. a protease naturally occurring in the host cell, or an externally added protease. For example, the externally added protease may be an IgA protease (cf.
  • the nucleic acid fusion comprises (iv) a portion encoding a transmembrane linker which is required for the presentation of the passenger polypeptide (ii) on the outer surface of the outer membrane of the host cell. Further, the nucleic acid fusion comprises (v) a transporter domain of an autotransporter.
  • transmembrane linker domain is used which is homologous with regard to the autotransporter, i.e. the transmembrane linker domain is encoded by a nucleic acid portion directly 5′ to the autotransporter domain.
  • the length of the transmembrane linker is preferably 30-160 amino acids.
  • the length of the recombinant polypeptide to be displayed i.e. the passenger polypeptide is preferably in the range of from 5-3000 amino acids, more preferably in the range from 10-1500 amino acids.
  • the transporter domain of the autotransporter according to the invention can be any transporter domain of an autotransporter and is preferably capable of forming a ⁇ -barrel structure.
  • a detailed description of the ⁇ -barrel structure and preferred examples of ⁇ -barrel autotransporters are disclosed in WO97/35022 incorporated herein by reference.
  • the transporter domain of the autotransporter may be selected from the E. coli AIDA-I protein, the Shigella flexneri Sep A protein, the Shigella flexneri IcsA protein, the E. coli Tsh protein, the Serratia marcescens Ssp protein, the Helicobacter mustelae Hsr protein, the Bordetella ssp.
  • Prn protein the Haemophilus influenzae Hap protein, the Bordetalla pertussis Brk A protein, the Helicobacter pylori Vac A protein, the surface protein SpaP, rOmpB or SIpT from Rickettsia, the IgA protease from Neisseria or Haemophilus and variants thereof.
  • the transporter domain of the autotransporter is the E. coli AIDA-I protein or a variant thereof, such as e.g. described by Niewert U., Frey A., Voss T., Le Bouguen C., Baljer G., Franke S., Schmidt M A.
  • the AIDA Autotransporter System is Associated with F18 and Stx2e in Escherichia coli Isolates from Pigs Diagnosed with Edema Disease and Postweaning Diarrhea. Clin. Diagn. Lab. Immunol. Jan. 2001, 8(1):143-149;9.
  • Variants of the above indicated autotransporter sequences can e.g. be obtained by altering the amino acid sequence in the loop structures of the ⁇ -barrel not participating in the transmembrane portions.
  • the nucleic acid portions coding for the surface loops can be deleted completely.
  • conserved amino exchanges i.e. the exchange of an hydrophilic by another hydrophilic amino acid or/and the exchange of a hydrophobic by another hydrophobic amino acid may take place.
  • a variant has a sequence identity of at least 50% and particularly at least 70% on the amino acid level to the respective native sequence of the autotransporter domain at least in the range of the ⁇ -sheets.
  • a further aspect of the present invention relates to a host cell displaying a functional recombinant polypeptide on the surface thereof wherein the recombinant polypeptide contains a prosthetic group and wherein the recombinant polypeptide is preferably displayed by the transporter domain of an autotransporter.
  • the polypeptide may be selected from ferredoxins, e.g. adrenodoxin, P450 reductases, cytochrome b5, P450 enzymes, flavoproteins, e.g. oxidoreductases, dehydrogenases or oxidases, especially sugar oxidases, such as pyranose oxidase (FIG. 15), and any combinations thereof.
  • Still a further embodiment of the present invention is a host cell displaying a functional recombinant polypeptide on the surface thereof wherein the recombinant polypeptide is a multimeric polypeptide containing at least two polypeptide subunits and wherein at least one subunit of the multimeric polypeptide is preferably displayed by the transporter domain of an autotransporter.
  • the invention relates to a membrane preparation which is derived from a host cell as described above wherein this membrane preparation displays a functional recombinant polypeptide containing a prosthetic group and/or being a multimeric polypeptide.
  • the method according to the invention and the host cells according to the invention can be used for a variety of different applications, e.g. as whole cell biofactories or membrane preparation biofactories for chemical synthesis procedures, e.g. for the synthesis of organic substances selected from enzyme substrates, drugs, hormones, starting materials and intermediates for syntheses procedures and chiral substances (cf. Roberts, Chemistry and Biology 6 (1999), R269-R272).
  • the cell or the membrane preparation of the invention may be used for a directed evolution procedure, e.g. for the development of new biocatalysts for the application in organic syntheses.
  • libraries of variants produced by site-specific or random mutagenesis of ferredoxins, in particular Adx are examined in view of the function of individual amino acids e.g. during electron transfer.
  • libraries of variants of P450 enzymes or flavoproteins are examined in view of the role of defined amino acids during certain functions, in particular catalytical functions.
  • these particular embodiments concern the production of variants of proteins and/or enzymes and the production of libraries with variants of proteins and/or enyzmes, respectively, which carry a prosthetic group or are coenzymes etc. or multimers etc. and which are screened in view of a certain characteristic, i.e. one or optionally several variants fulfilling this desired characteristic perfectly are selected.
  • a certain characteristic i.e. one or optionally several variants fulfilling this desired characteristic perfectly are selected.
  • the cell is selected, too, and carries the nucleic acid coding the variant.
  • both the amino acid sequence and the structural information of the variant can be determined via the nucleic acid sequence.
  • the characteristics in question are particularly enzyme inhibiting, catalytical, toxin degrading, synthesizing, therapeutical etc. characteristics.
  • the host cell or the membrane preparation may be used as an assay system for a screening procedure, e.g. for identifying modulators (activators or inhibitors) of displayed polypeptides such as P450 enzymes or flavoproteins which may be used as potential therapeutic agents.
  • the screening procedure may also be used to identify variants of displayed polypeptides having predetermined desired characteristics.
  • libraries of modulators and/or libraries of displayed polypeptides may be used.
  • the host cells or membrane preparations derived therefrom may be used as a system for toxicity monitoring and/or degrading toxic substances in the environment, in the laboratory or in biological, e.g. human, animal, or non-biological systems.
  • An essential advantage of applying the host cells and membranes according to the invention is enabling correct folding and biological activity of proteins or protein complexes, e.g. P450 enzymes or flavoproteins, which require a membrane environment.
  • a reconstitution as previously considered to be necessary is no longer required.
  • the production steps of a functional biocatalytic system are simplified and an increased stability of the system per se is obtained.
  • AdR and P450 enzymes e.g. CYP11 B1 or CYP11A1 externally.
  • AdR, Adx and P450 FIG. 5, FIG. 9 and FIG. 14
  • the membrane environment is provided by the bacterial cell so that adding an artificial membrane or further detergents or a reconstitution as in previously used systems is not necessary.
  • low amounts of detergent can be added to the host cell and/or membrane preparation according to the invention.
  • These functional complexes can serve as carrier to target e.g.
  • the method according to the invention allows for an efficient expression of passenger proteins on the surface of host cells, particularly E. coli or other gram-negative bacterial cells up to 100 000 or more molecules per cell by using a liquid medium of the following composition: 5 g/l to 20 g/l, preferably about 10 g/l trypton, 2 g/l to 10 g/l, preferably about 5 g/l yeast extract, 5 g/l to 20 g/l, in particular about 10 g/l NaCl and the remaning part water.
  • the medium should possibly contain as little as possible divalent cations, thus preferably Aqua bidest or highly purified water, e.g. Millipore water is used.
  • the liquid medium contains in addition preferably EDTA in a concentration of 2 ⁇ M to 20 ⁇ M, in particular 10 ⁇ M. Moreover, it contains preferably reducing reagents, such as 2-mercapto ethanol or dithiotreitol or dithioerythritol in a preferred concentration of 2 mM to 20 mM. The reducing reagents favour a non-folded structure of the polypeptide during transport.
  • the liquid medium can further contain additional.
  • C-sources preferably glucose, e.g. in an amount of up to 10 g/l, in order to favour secretion i.e. transfer of the passenger to the surrounding medium. For surface display preferably no additional C-source is added.
  • host cells which carry a ferredoxin e.g. Adx or/and AdR or/and P450 reductase or/and cytochrome b5 or/and P450 enzymes or/and one of the peptides described in the following on their surface in a way that at least one polypeptide chain is brought to the surface with the help of an autotransporter and the prosthetic groups are inserted afterwards in replacement of previously used microsomal systems or systems reconstituted with the help of artificial or natural membranes or membrane parts.
  • a ferredoxin e.g. Adx or/and AdR or/and P450 reductase or/and cytochrome b5 or/and P450 enzymes or/and one of the peptides described in the following on their surface in a way that at least one polypeptide chain is brought to the surface with the help of an autotransporter and the prosthetic groups are inserted afterwards in replacement of previously used microsomal systems or systems reconstituted with the help of artificial or natural membranes or membrane parts.
  • the passenger polypeptides are peptides or proteins selected from the group of drug metabolizing enzymes, such as CYP1A2 involved in the activation of aromatic amine carcinogenes, heterocyclic arylamine promutagenes derived from food pyrolysates and aflatoxin B1 (Gallagher EP, Wienkers LC, Stapleton PL, Kunze KL, Eaton DL., Role of human microsomal and human complementary DNA-expressed cytochromes P4501A2 and P4503A4 in the bioactivation of aflatoxin B1. Cancer Res. Jan.
  • CYP2E1 capable of activating the procarcinogenes N-nitrosodimethylamine and N-nitrosodiethylamin and metabolizes the procarcinogenes benzene, styrene, carbon tetrachloride, chloroform (Yoo JS, Ishazaki H, Yang CS., Roles of cytochrome P45011E1 in the dealkylation and denitrosation of N-nitrosodimethylamine and N-nitrosodiethylamine in rat liver microsomes. Carcinogenesis.
  • passenger peptides are peptides from the group of steroid biosynthesis enyzmes, such as CYP11B1 involved in the formation of cortisol and aldosterone (Bernhardt R., Cytochrome P450: structure, function and generation of reactive oxygen species. Rev. Physiol. Biochem. Pharmacol.
  • peptides from the group of metal ion containing enzymes in particular Zn-containing enzymes, such as Zn-containing lactamases, carboanhydrase and alcohol dehydrogenase, Mg-containing enzymes, such as hexokinase, glucose-6-phosphatase or pyruvate kinase, Ca-containing enzymes, Fe-containing enzymes such as cytochrome oxidase, catalase, peroxidase or Ni-containing enzymes, such as urease catalyzing the hydrolization of urea to form ammonia and carbondioxide, or the hydrolization of urea-like compounds (Mobley H L, Island M D, Hausinger R P., Molecular biology of microbial ureases.
  • Zn-containing enzymes such as Zn-containing lactamases, carboanhydrase and alcohol dehydrogenase
  • Mg-containing enzymes such as hexokinase, glucose-6-phosphat
  • metal ion containing enzymes are Cu-containing enzymes, such as cytochrome-oxidase, Mn-containing enzymes, such as arginase and ribonucleotide reductase, Mo-containing enzymes, such as dinitrogenase and Se-containing enzymes, such as glutathione peroxidase.
  • the recombinant polypeptide to be displayed i.e. the passenger polypeptides are peptides or proteins from the group of flavoproteins, e.g. oxidoreductases, dehydrogenases or oxidases, in particular sugar oxidases, such as pyranose oxidase.
  • host cells which carry a sugar oxidase, e.g. pyranose oxidase, on their surface by bringing the polypeptide chain of the sugar oxidase to the surface with the help of an autotransporter and subsequently adding the prosthetic group FAD.
  • this can be achieved by adding purified FAD in a buffer solution to the host cells displaying the sugar oxidase polypeptide chain, wherein the buffer solution contains FAD in excess, 1 mM dithiothreitol, 0.1 mM EDTA, 17% glycerol (v/v), 0.1 M guanidine/HCI and 0.1 M HEPES, pH 7.5.
  • the procedure described above for the insertion of the prosthetic group can also be transferred to those flavoproteins that are described to be homotetramers, homodimers, etc.
  • the host cells or membrane preparations derived therefrom may be used as systems for the synthesis of sugars, building blocks, fine chemicals, basic chemicals and chiral compounds.
  • host cells and/or membrane preparations are provided, which carry at least one P450 enzyme, on their surface in a way that at least one polypeptide chain is brought to the surface with the help of an autotransporter and the prosthetic groups are inserted afterwards in replacement of previously used microsomal systems or systems reconstituted with the help of artificial or natural membranes or membrane parts.
  • the P450 enzymes are hepatic P450 enzymes, particularly P450 3A4, 2D6, 2C9 and 2C19.
  • the host cells and/or preparations according to the invention are preferably used sequentially for testing the enzyme inhibition of P450 enzymes.
  • the so-called lead identification whether the new drug lead structure to be tested could possibly have side-effects or lead to the so-called drug-drug interaction.
  • FIG. 1 Adx dependent reactions of CYP1A1 and CYP1B1.
  • the electron transfer activity of surface-presented Adx was analyzed in reconstituted systems containing the natural final electron acceptors CYP11A1 and CYP11B1 catalyzing the indicated chemical reactions.
  • FIG. 2 (A) Nucleotide and amino acid sequence of bovine adrenodoxin, devoid of the mitochondrial target sequence as used in this study.
  • the environment of the fusion sites are given as sequences. Signal peptidase and trypsin cleavage sites are indicated.
  • FIG. 4 HPLC chromatograms of CYP11A1 and CYP11B1-dependent substrate conversion. Chromatograms were obtained from extracted samples with E. coli cells containing pAT-Adx04 before reconstitution (A, C) and after reconstitution (B, D) of the iron-sulfur cluster in surface displayed adrenodoxin.
  • CYP11A1 reactions represent 1) cortisol (internal standard), 2) pregnenolone (product) and 3) cholesterone (substrate), in CYP11B1 reactions (C, D) 4) cortisol (internal standard), 5) corticosterone (product) and 6) deoxycorticosterone (substrate).
  • Steroids were analyzed for A and B with an isocratic solvent system of acetonitril/isopropanol (15:1) and for B and C with an isocratic solvent system of 50% acetonitril.
  • FIG. 5 Schematic representation of Adx surface display by the autotransporter pathway in E. coli.
  • FIG. 6 SDS-Page (A) and Western blot (B) of outer membrane preparations from E. coli UT2300 (lane 1) and E. coli UT2300 pJJ004 (lane 2). Before outer membrane were prepared, whole cells were digested with trypsin (+) or not ( ⁇ ). Western blot was performed with an Adx specific antibody.
  • FIG. 8 Western blot of outer membrane preparations from E. coli UT2300 pJJ004 treated with different sample buffers before SDS-Gel separation.
  • SB + sample buffer contained mercaptoethanol
  • red ⁇ sample buffer without mercaptoethanol.
  • Western blot was performed with an Adx specific antibody.
  • FIG. 9 Schematic representation of functional ADX dimers on the surface of E. coli.
  • FIG. 10 SDS-PAGE (A) and Western blot analysis of outer membrane preparations from E. coli UT5600 pJJ004. Whole cells were either digested with trypsin (lane 2) or not (1) before outer membranes were prepared. Before being applied to SDS-PAGE samples were boiled in sample buffer without 2-mercaptoethanol. The sizes of the molecular weight marker bands are indicated. Natural outer membrane proteins OmpF/C and OmpA are marked. The Adx specific antibody used for detection has been described previously (35).
  • FIG. 11 (A) Western blot analysis of supernatant proteins from OmpT + E. coli UT2300. Before applied to SDS-PAGE samples were boiled with (lane 1) or without 2-mercaptoethanol (2). The sizes of the molecular weight marker proteins are indicated.
  • Adx Molecular weight determination of free, recombinant Adx.
  • the size of purified Adx molecules, not connected to the autotransporter domains was determined by the use of size exclusion chromatography.
  • the proteins indicated in the plot (circles with grey fill color) were used to generate a standard curve.
  • the retention volume of Adx is shown as a circle with white fill color and the extrapolated MW is given in brackets.
  • FIG. 12 Released dimeric Adx on the surface of E. coli UT2300 pJJ004.
  • A SDS-PAGE
  • B Western blot analysis
  • whole cells were treated with trypsin (+) or not ( ⁇ ).
  • buffer without 2-mercaptoethanol was used for sample preparation. The migration of the molecular weight maker proteins is indicated in kilodaltons.
  • FIG. 13 Schematic view of the whole cell steroid conversion by autotransporter mediated surface display of dimeric Adx in E. coli.
  • FIG. 14 Amino acid sequence of pyranose oxidase from Coriolusolor as can be used in this study.
  • E. coli UT5600 F ⁇ ara14 leuB6 azi-6 lacY1 proC14 tsx-67 entA403 trpE38 rfbD1 rpsL109 xyl-5 mtl-1 thi1, ⁇ ompT-fepC266) was used for the expression of autotransporter fusion proteins (42).
  • E. coli UT5600 F ⁇ ara14 leuB6 azi-6 lacY1 proC14 tsx-67 entA403 trpE38 rfbD1 rpsL109 xyl-5 mtl-1 thi1, ⁇ ompT-fepC266) was used for the expression of autotransporter fusion proteins (42).
  • E. coli UT5600 F ⁇ ara14 leuB6 azi-6 lacY1 proC14 tsx-67 entA403 trpE38 rfbD1 rpsL109 xyl-5 m
  • coli TOP10 F ⁇ mcrA ⁇ (mrr-hsdRMS-mcrBC) ⁇ 80lacZ ⁇ M15 ⁇ lacX74 deoR recA1 araD139 ⁇ (ara-leu) 7697 galU galK rpsL (Str R ) endA1 nupG
  • Plasmid pJM007 (15), encoding the AIDA-I autotransporter and plasmid pKKHCAdx (43), encoding bovine adrenodoxin have been described elsewhere. Bacteria were routinely grown at 37° C.
  • EDTA was added to a final concentration of 10 ⁇ M and ⁇ -mercaptoethanol was added to a final concentration of 10 mM.
  • the Adx gene was amplified by PCR from plasmid pKKHCAdx using oligonucleotide primers JJ3 (5′-ccgctcgagggcagctcagaagataaaataacagtc-3′) and JJ4 (5′-ggggtaccttctatctttgaggagttcatg-3′).
  • JJ3 5′-ccgctcgagggcagctcagaagataaaataacagtc-3′
  • JJ4 5′-ggggtaccttctatctttgaggagttcatg-3′.
  • the PCR product was inserted into vector pTOPO10 and recleaved with Xhol and Kpnl.
  • the restriction fragment was ligated to pJM7, restricted with the same enzymes. This yields an in-frame fusion of Adx with the AIDA-I autotransporter, under the control
  • E. coli cells were grown overnight and 1 ml of the overnight culture was used to inoculate 20 ml LB medium. Cells were cultured at 37° C. with vigorous shaking (200 rpm) for about 5 h until an OD 578 of 0.7 was reached. After harvesting and washing with phosphate-buffered saline (PBS), outer membranes were prepared according to the rapid isolation method of Hantke (44). For whole cell protease-treatment, E. coli cells were harvested, washed and resuspended in 5 ml PBS. Trypsin was added to a final concentration of 50 mg liter ⁇ 1 and cells were incubated for 5 min at 37° C. Digestion was stopped by washing the cells three times with PBS containing 10% fetal calf serum (FCS) and outer membranes were prepared as described above.
  • PBS phosphate-buffered saline
  • Outer membrane isolates were diluted 1:2 with 2 ⁇ sample buffer (100 mM Tris/HCl, pH 6.8, 4% SDS, 0.2% bromphenol blue, 20% glycerol), either with (reducing) or without 2-mercaptoethanol (non-reducing conditions), boiled for 20 min and analyzed on 12.5% SDS-PAGE. Proteins were visualized with Coomassie brilliant blue with prestained molecular weight protein markers (Bio-Rad, Ober, Germany). For Western blot analysis, gels were electroblotted onto polyvinylidene-difluoride (PVDF) membranes and blotted membranes were blocked in PBS with 3% FCS overnight.
  • PVDF polyvinylidene-difluoride
  • membranes were incubated with primary anti-Adx antibody, diluted 1:500 in PBS with 3% FCS for 3 hours. Prior to addition of the secondary antibody, immunoblots were rinsed three times with PBS. Antigen-antibody conjugates were visualized by reaction with horseradish peroxidase-linked goat anti-rabbit IgG secondary antibody (Sigma, Deisenhofen, Germany), dilutes 1:1000 in PBS. Color reaction was achieved by adding a solution consisting of 2 ml 4-chlor-1-naphtol (3 mg/ml ethanol), 25 ml PBS and 10 ⁇ l H 2 O 2 (30%).
  • the bacterial suspension (4 ml) was supplemented with 1 mM ⁇ -mercaptoethanol and 0.2 mM ferrous ammonium sulfate and was slowly titrated with 100 ml of a solution containing 100 mM Li 2 S and 2 mM DTT (45).
  • Adx Recombinant adrenodoxin
  • AdR adrenodoxin reductase
  • Isolation of CYP11A1 and CYP11B1 from bovine adrenals was performed according to Akhrem et al. (49) with slight modifications.
  • adrenodoxin The biological electron transfer function of adrenodoxin was detected in adrenodoxin-dependent reactions containing its natural effector enzymes, cytochromes CYP11A1 and CYP11B1.
  • the cholesterol side chain cleavage activity of cytochrome CYP11A1 was assayed in a reconstituted system catalyzing the conversion of cholesterol to pregnenolone. Assays were performed at 37° C. in 50 mM potassium phosphate (pH 7.4), 0.1% Tween 20 and contained 100 ⁇ l E. coli cells, 0.5 ⁇ M adrenodoxin reductase, 0.4 ⁇ M CYP11A1, 400 ⁇ M cholesterol and a NADPH regenerating system.
  • the steroids were converted into their corresponding 3-one-4en forms by the addition of 2 units/ml cholesterol oxidase, extracted, and analyzed by reversed phase HPLC.
  • Substrate conversion from deoxycorticosterone to corticosterone in cytochrome CYP11B1 assays were performed at 37° C. in 50 mM potassium phosphate (pH.7.4), 0.1% Tween 20 and consisted of 100 ⁇ l E. coli cells, 0.5 ⁇ M adrenodoxin reductase, 0.2 ⁇ M CYP11B1, 400 ⁇ M deoxycorticosterone and a NADPH regenerating system.
  • EPR measurements were carried out on a Bruker ESP300E spectrometer at ⁇ 163° C.
  • Cell samples in 50 mM potassium phosphate (pH 7.4) were dithionite-reduced under anaerobic conditions and frozen in liquid nitrogen.
  • the coding region of bovine adrenodoxin (Adx) was PCR amplified.
  • the PCR primers used added a Xhol site at the 5′ end and a Kpnl site at the 3′ end of the Adx encoding region.
  • an Adx gene was used as PCR template that was devoid of the mitochondrial targeting sequences (32).
  • the amino acid and the nucleotide sequence of the resulting PCR product, which was confirmed by dideoxy sequencing is shown in FIG. 2 a.
  • plasmid pJM7 was cleaved with Xhol and Kpnl.
  • pJM7 is a pBR322-derived high copy number plasmid that directs the expression of a choleratoxin ⁇ -AIDA-I fusion protein under control of the constitutive P TK promoter (15,23). Cleavage with Xhol and Kpnl resulted in the deletion of the choleratoxin ⁇ (CTB) encoding DNA region.
  • CTB choleratoxin ⁇
  • plasmid pJJ004 Insertion of the cleaved Adx PCR fragment yielded plasmid pJJ004, which encoded a fusion protein consisting of the signal peptide of CTB, Adx, and the AIDA-I autotransporter region, including a linker region, which proved to be sufficient for full surface access (FIG. 2 b ). Due to the ligation procedure the artificial construct still contains seven amino acids of mature CTB. Based on the predicted molecular mass of 65.9 kDa, the fusion protein was termed FP66. Export of FP66 was investigated in E. coli.
  • E. coli host strains possess an outer membrane protease (OmpT) that catalyzes the sequence specific release of surface-exposed proteins (33).
  • OmpT outer membrane protease
  • E. coli UT5600 (ompT) proved to be suitable to prevent cleavage of surface-exposed autotransporter fusion proteins (23,34). Therefore pJJ004 was transformed into E. coli UT5600 and the expression of FP66 was monitored by SDS-PAGE and immunoblotting of outer membrane protein preparations. As shown in FIG.
  • FP66 could be easily detected by Coomassie brilliant blue staining of outer membrane proteins. Expression was almost at the same level as expression of the natural outer membrane proteins OmpA and OmpF/C. Neither growth rate nor optical density reached in the stationary phase of E. coli UT5600 grown in liquid medium was decreased by the expression of FP66 (not shown). Electrophoretic mobility of the Adx-AIDA ⁇ -fusion protein was in perfect agreement with the predicted molecular mass of 65.9 kDa. Finally, the identity of FP66 was confirmed by Western blot analysis using an Adx specific polyclonal rabbit antibody (35) (FIG. 3 b ). The application of non-reducing conditions resulted in faint bands at molecular weights that corresponded to multimers of the fusion protein.
  • Predicted trypsin cleavage sites that are located closer to the C-terminus of the transporter are protected from trypsin access by membrane topology.
  • SDS-PAGE and subsequent staining with Coomassie blue external trypsin addition resulted in the disappearance of the full-size-fusion protein and generated two lower-molecular-weight products (FIG. 3 a ).
  • One of them corresponds to the 37.1 kDa trypsin resistant autotransporter core.
  • the second digestion product (core 2) has a larger molecular weight of around 45 kDa. Therefore it must result from trypsin cleavage within the Adx passenger domain. Folding subsequent to processing could hinder trypsin access to the linker region and therefore prevent further degradation.
  • Bovine Adx is known to contain several consensus trypsin cleavage sites. Obviously in our experiments, there was a preference for the trypsin cleavage of distinct sequence, as beside the 45 kDa core 2, there was no other prominent digestion product detectable.
  • Adx displayed on the E. coli surface by the autotransporter pathway is biologically active and can transfer electrons to the P450 enzymes CYP11A1 and CYP11B1.
  • the number of active Adx molecules after reconstitution on the surface of E. coli could be estimated by the use of the specific substrate conversion rate in the CYP11A1 assay. For this purpose the enzyme assay was performed without E. coli cells but with different concentrations of purified holo-Adx.
  • Adx molecules per cell An apparent number of 1.8 ⁇ 10 5 functional Adx molecules per cell indicated optimal conditions for refolding of the peptide around the 12Fe-2S] center at 22° C. A preceding heat denaturation of surface-displayed apo-Adx was obviously not necessary for successful cluster incorporation.
  • bovine adrenodoxin was expressed on the E. coli cell surface by the autotransporter pathway.
  • the expression rate was in the same order of magnitude as the expression of natural outer membrane proteins OmpF/C or OmpA without disturbing outer membrane integrity or reducing cell growth (FIG. 3 a ).
  • Adx passenger molecules transported to the cell surface by the autotransporter initially did not contain an iron-sulfur cluster. This fits the concept of the autotransporter secretion mechanism (15,17,20). Accordingly, the C-terminus of this secreted proteins forms a porin-like structure, a so-called ⁇ -barrel in the outer membrane and proteins with stable and extended three dimensional structures cannot pass this gate.
  • Activity could easily be quantified by determining Adx depending product formation of either pregnenolone or corticosterone by HPLC.
  • Adx activity By calibrating Adx activity in the substrate formation assay by purified holo-Adx, the apparent number of active Adx molecules could be determined as more than 100.000 molecules/cell.
  • the high copy number of recombinant outer membrane proteins is at the same level as it has been reported for natural E. coli outer membrane protein OmpA (39). High expression of recombinant and chemically reconstituted, active Adx, had no influence on the viability of E. coli . Reconstitution at higher temperature in order to get easier unfolding of the Apo-Adx peptide chain did not result in a better [2Fe-2S] cluster incorporation.
  • the role of distinct amino acids in the electron transfer through Adx or in the interaction with its redox partners can be studied either by random or by rational variation of the protein, without the need of mutant enzyme purification.
  • heme containing P450 enzymes are important, as they are involved in the syntheses of a wide variety of valuable products but also in the degradation of numerous toxic compounds (41).
  • autotransporter mediated surface display of e.g. P450 enzymes could open a new dimension in developing whole cell factories.
  • Adx dimers cleaved off from the transporter domain remained associated to the outer membrane and were not released to the supernatant. It has been shown earlier that Adx not only forms dimers but also forms tetramers. By this view it might be possible that Adx dimers that were released by OmpT form hybrid tetramers with Adx molecules that are still connected to the surface by the autotransporter domain. It cannot be excluded that some amount of Adx was released to the supernatant, but remained below the detection limit. At this point it seems worth emphazising that incomplete processing of autotransporter proteins by OmpT is not common.
  • a protein band with an apparent molecular weight corresponding to a tetramer of free Adx molecules could not be ascertained.
  • External addition of trypsin resulted in the complete disappearence of the full-size-fusion protein as well as the putative Adx dimer (FIG. 6), underlining the surface location of both forms of the protein.
  • Plasmid pJJ004 was propagated in E. coli strains UT5600 (F ⁇ ara14 leuB6 azi-6 lacY1 proC14 tsx-67 entA403 trpE38 rfbDI rpsLI09 xyl-5 mt1-l thi1, ⁇ ompT-fepC266) and UT2300 (F ⁇ ara14 leuB6 azi-6 lacY1 proC14 tsx-67 entA403 trpE38 rfbDI rpsLI09 xyl-5 mtl-l thil) (42).
  • E. coli UT2300 releases surface-exposed passenger proteins, whereas E. coli UT5600 is not able to form the outer membrane protease T (OmpT), resulting in the surface display of passenger proteins that are translocated by the autotransporter pathway (1 5).
  • Cells were routinely grown at 37° C. in Luria-Bertani (LB) broth containing 100 mg l ⁇ 1 ampicillin, 10 ⁇ M EDTA and 10 mM 2-mercaptoethanol.
  • LB Luria-Bertani
  • glucose was added up to 5% and to achieve a real low metal ion supplementation, growth medium was prepared with tap water, when indicated.
  • E. coli cells were grown overnight in LB medium and 1 ml was used to inoculate a 20 ml culture. Cultivation was at 37° C. with vigorous shaking (200 rpm) until an OD 578 of 0.7 was reached (mid log phase). Cells were harvested by centrifugation and after washing with phosphate-buffered saline (PBS), outer membranes were prepared according to the rapid isolation method of Hantke (44). For whole cell protease-treatment, E. coli cells were harvested, washed and resuspended in 5 ml PBS. Trypsin was added to a final concentration of 50 mg l ⁇ 1 and cells were incubated for 5 min at 37° C.
  • PBS phosphate-buffered saline
  • the Adx dependent cholesterol side chain cleavage activity of cytochrome CYP11A1 was assayed in an assembled system catalyzing the conversion of cholesterol to pregnenolone (68,67). Assays were performed for 30 min at 37° C. in 50 mM potassium phosphate (pH 7.4) and contained 100 ⁇ l E. coli cells, 0.5 ⁇ M adrenodoxin reductase, 0.4 ⁇ M CYP11A1, 400 ⁇ M cholesterol, 60 ⁇ M NADPH and a NADPH regenerating system. After the side chain cleavage reaction, the steroids were converted into their corresponding 3-one-4-en forms by the addition of 2 Units/ml cholesterol oxidase and extracted with chloroform.
  • the efficient surface display of bovine adrenodoxin in Escherichia coli by the autotransporter pathway can be facilitated by the construction of an artificial gene.
  • This gene encodes a fusion protein, consisting of Adx, a signal peptide and the translocation unit of the Adhesin involved in diffuse adhesion (AIDA-I), a natural autotransporter protein of E. coli (51) (FIG. 2B).
  • the translocation unit contains the C terminal ⁇ -barrel and a linker region, that is necessary for full surface exposure of the passenger domain (15).
  • the artificial gene had to be expressed in an outer membrane protease T (ompT) mutant of E. coli .
  • OmpT cleaves proteins in the cell envelope of E. coli (42) and the linker region of AIDA-I contains a consensus cleavage site (R/V) (15).
  • R/V consensus cleavage site
  • the full size fusion protein (FP66) could be easily detected by Coomassie staining of SDS polyacrylamide gels, when outer membrane preparations were applied (FIG. 10).
  • the fusion protein could also be identified by labeling with an Adx specific antibody in Western blot experiments (FIG. 10B).
  • plasmid pJJ004 encoding FP66 was expressed in E. coli strain UT2300.
  • This strain isogenic to E. coli UT5600 with the exception that it is able to form an active outer membrane protease T (ompT + ) (1).
  • supernatants of E. coli UT2300 pJJ004 we could detect a protein that was labeled by the Adx specific antibody (FIG. 11).
  • outer membrane preparations from ompT ⁇ UT2300 were also analysed for the fate of passenger Adx and full size fusion protein FP66.
  • processing of FP66 by OmpT was rather limited.
  • the majority of fusion protein remained in its initial size within the outer membrane. This can explain, why we found only weak amounts of released passenger Adx in the supernatant.
  • a protein was co-purified with the outer membrane that had an identical size with the supernatant Adx dimers and that was labeled by the Adx specific antibody. This means, that on one hand OmpT has only weakly processed FP66 and on the other, that the majority of released Adx passenger domains remained for some reasons associated with the outer membrane.
  • bovine Adx was purified after recombinant expression in E. coli and subjected to size exclusion chromatography. As can be seen in FIG. 11B, the apparent molecular weight of free Adx determined by this method (24 kDa) was quite close to the calculated•molecular weight of a dimeric molecule (28.8 kDa) indicating, that indeed free Adx also forms dimers.
  • Example 1 we showed that the iron sulfur-cluster can be effectively incorporated into apo-Adx displayed on the E. coli coli surface.
  • Adx devoid of the 2[Fe—S] cluster is biologically not active. Due to the autotransporter secretion mechanism, however, Adx can only be transported to the surface in an unfolded state as apo-Adx without prosthetic group.
  • Iron-sulfur cluster incorporation appeared to be best under anaerobic conditions, when LiS 2 was added dropwise to Adx-expressing cells in a ferrous ammonium sulfate buffer at room temperature. By this procedure iron sulfur clusters were formed and immediately incorporated into apo-Adx displayed at the bacterial surface.
  • E. coli UT5600 pJJ004 as well as E. coli UT2300 pJJ004 were treated this way and outer membranes were prepared and analyzed by SDS-PAGE and Western blotting. The results were identical to those obtained before chemical reconstitution (FIG. 10, FIG. 12).
  • E. coli UT5600 pJJ004 multimers of the full size fusion protein were detectable, and in E. coli UT2300 pJJ004 the majority of Adx, released from the transporter unit, remained associated to the surface as dimers (not shown). This indicates, that dimer formation is not a special property of apo-Adx, devoid of the iron-sulfur cluster and that iron-sulfur cluster incorporation does not affect dimer formation.
  • Adx molecules displayed on the surface As the number of Adx molecules displayed on the surface has been determined to be more than 10 5 (see Example 1), they can be assumed to obtain sufficient vicinity. Because they should be freely motile, the ⁇ -barrels within the outer membrane cannot offer much resistance against this affinity of passengers. To our knowledge, this is the first report on the functional, passenger driven, dimerization of a protein on the surface of E. coli.
  • Adx molecules are initially expressed as monomers, from monomeric genes. Dimerization is self-directed and does not require any connection in between of Adx monomers by a linker peptide, as applied for so-called single chain antibodies (55). Therefore the autotransporter mediated surface display of single polypeptides that can dimerize or multimerize on the surface offer new dimensions in the field of biotechnology applications as e.g. antibody technology.
  • the autotransporter ⁇ -barrel is structurally related to the ⁇ -barrel of the so-called porins, channels for small hydrophilic molecules within the outer membrane.
  • the porins like OmpF, have been shown to form trimers and monomers are assumed to be thermodynamically unstable (65).
  • Adx thermodynamically unstable
  • a mixed scenario in which e.g. the autotransporter ⁇ -barrel propagates approximation of the passenger domains to finally form stable dimers by self-contact, might be conceivable. But up to now there is no experimental evidence.
  • Bovine Adx contains five cysteines of which four are involved in the iron-sulfur cluster binding. Theoretically the fifth cysteine could be available for disulfide bonding. From the crystal data (50), however, this can be excluded.
  • the autotransporter used in this Adx-autotransporter fusion contained no cysteines at all (15). Therefore in our experiments, it is very unlikely, that dimerization results from disulfide bonding. This means, that dimer formation is due to an interaction or bonding that is stable enough to withstand 20 min of boiling and is resolved by the addition of ⁇ -mercaptoethanol but is no disulfide bond.
  • glycophorin A 66
  • ⁇ -glutamyltranspeptidase 59
  • glycophorin A a methionine residue plays the important role for dimerization by hydrophobic interactions.
  • ⁇ -glutamyltranspeptidase strong ionic and/or hydrophobic interactions were discussed, whereas in both cases disulfide bonds seem not to be involved.
  • CYP11A1 catalyzes the first and rate limiting step in steroid hormones biosynthesis, the cholesterol side chain cleavage of cholestrol to form pregnenolone; wide substrate specificity with respect to the length of the side chain and the position of the hydroxygroup (Usanov, et al. 1990).
  • CYP11B1 involved in biosynthesis of the main corticosteroid cortisol catalyzes the 11 ⁇ -hydroxylation of 11-deoxycortisol to form cortisol (Okamoto and Nonaka 1990).
  • CYP11B2 involved in biosynthesis of the main minaralcorticoid aldosterone, catalyzes the 11 ⁇ -hydroxylation of deoxycorticosterone to cortisone and the following 18-oxidation to form aldosterone (Okamoto and Nonaka 1992).
  • CYP12A1 involved in steroid metabolism of insect cells; metabolizes a number of insecticides and several xenobiotics; catalyzes different types of chemical reactions such as hydroxylations, epoxidation, N- demethylation, O-alkylation, desulfuration, and oxidative ester cleavage (Guzov, et al. 1998).
  • CYP24 initiates the degradation of 1,25-dihydroxyvitamin D3, the physiologically active form of vitamin D3, by hydroxylation of the side chain (Chen, et al. 1993).
  • CYP27 involved in the bile acid biosynthetic pathways catalyzing 27- hydroxylation and multiple oxidation reactions at the C-27 atom of steroids and is involved in the activation of vitamin D, catalyzes 24-, 25-, and 26(27)-hydroxylation reactions in Vitamine D derivatives (Cali and Russell 1991) (Guo, et al. 1993).

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